Phospholipase A 2 Metabolites Mediate Endothelin Stimulation of the Human Brain Natriuretic Peptide Promoter

Hypertension ◽  
2000 ◽  
Vol 36 (suppl_1) ◽  
pp. 721-721
Author(s):  
Quan He
Keyword(s):  
Gene Expression ◽  
Brain Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Promoter Activity ◽  
Luciferase Activity ◽  
Phospholipase A ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
P450 Monooxygenase ◽  
Stimulation Of

P155 Brain natriuretic peptide (BNP) gene expression accompanies cardiac hypertrophy and heart failure. The vasoconstrictor endothelin-1 (ET)may be involved in the development of these diseases. ET has also been shown to activate phospholipase A 2 (PLA 2 ). Thus we studied whether ET and PLA 2 metabolites regulate BNP gene expression. The hBNP promoter (-1818 to + 100) coupled to a luciferase reporter gene was transferred into neonatal ventricular myocytes (NVM),and luciferase activity was measured as an index of promoter activity. ET (10 -7 M)induced BNP mRNA in NVM as assessed by Northern blot. It also stimulated the hBNP promoter 4-fold vs control, an effect completely inhibited by actinomycin D. To test the involvement of different PLA 2 isoforms, transfected cells were treated with the Ca ++ -independent PLA 2 (iPLA 2 )inhibitor bromoenol lactone (BEL), the cytosolic PLA 2 inhibitor methyl arachidonyl fluorophosphonate, or the secretory PLA 2 inhibitor ONO-RS-082 prior to stimulation with ET. Only the iPLA 2 inhibitor BEL prevented ET-stimulated hBNP promoter activity. The PLA 2 metabolite lysophosphatidic acid (LPA) also activated the hBNP promoter (2.2-fold; n = 3), but lysophosphatidylcholine did not. To test whether arachidonic acid metabolites are involved in ET’s effect, cells were pretreated with either a lipoxygenase (LO), cyclooxygenase, or p450 monooxygenase inhibitor. Only the LO inhibitor baicalein prevented ET stimulation of the hBNP promoter. Finally, we studied the involvement of cis elements in ET-stimulated hBNP promoter activity. Deletion of BNP promoter sequences from -1818 to -408 and from -408 to -40 reduced ET’s effect by 54% and 78%, respectively. Moreover, ET-stimulated luciferase activity was reduced by 53% when the GATA element (at position -85 relative to the start site of transcription) was mutated. These data suggest that: 1) ET activates the hBNP promoter through a transcriptional mechanism; 2) LPA, perhaps generated by a BEL-sensitive iPLA 2 , is involved in ET’s effect; 3) a LO pathway may also mediate ET signaling; and 4) ET regulation of the hBNP promoter targets both distal and proximal cis elements, including GATA.

scholarly journals Isoproterenol and cAMP regulation of the human brain natriuretic peptide gene involves Src and Rac

2000 ◽  
Vol 278 (6) ◽  
pp. E1115-E1123 ◽  
Author(s):  
Quan He ◽  
Guiyun Wu ◽  
Margot C. Lapointe
Keyword(s):  
Brain Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Promoter Activity ◽  
Kinase Inhibitor ◽  
Regulatory Elements ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Dominant Negative Mutant ◽  
Luciferase Reporter Gene ◽  
Src Tyrosine Kinase

Brain natriuretic peptide (BNP) gene expression and chronic activation of the sympathetic nervous system are characteristics of the development of heart failure. We studied the role of the β-adrenergic signaling pathway in regulation of the human BNP (hBNP) promoter. An hBNP promoter (−1818 to +100) coupled to a luciferase reporter gene was transferred into neonatal cardiac myocytes, and luciferase activity was measured as an index of promoter activity. Isoproterenol (ISO), forskolin, and cAMP stimulated the promoter, and the β2-antagonist ICI 118,551 abrogated the effect of ISO. In contrast, the protein kinase A (PKA) inhibitor H-89 failed to block the action of cAMP and ISO. Pertussis toxin (PT), which inactivates Gαi, inhibited ISO- and cAMP-stimulated hBNP promoter activity. The Src tyrosine kinase inhibitor PP1 and a dominant-negative mutant of the small G protein Rac also abolished the effect of ISO and cAMP. Finally, we studied the involvement of M-CAT-like binding sites in basal and inducible regulation of the hBNP promoter. Mutation of these elements decreased basal and cAMP-induced activity. These data suggest that β-adrenergic regulation of hBNP is PKA independent, involves a Gαi-activated pathway, and targets regulatory elements in the proximal BNP promoter.

Regulation of the human brain natriuretic peptide gene by GATA-4

2002 ◽  
Vol 283 (1) ◽  
pp. E50-E57 ◽  
Author(s):  
Quan He ◽  
Mariela Mendez ◽  
Margot C. LaPointe
Keyword(s):  
Brain Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Promoter Activity ◽  
Nuclear Protein ◽  
Ventricular Myocytes ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Specific Expression

Brain natriuretic peptide (BNP) is a cardiac hormone constitutively expressed in the adult heart. We previously showed that the human BNP (hBNP) proximal promoter region from −127 to −40 confers myocyte-specific expression. The proximal hBNP promoter contains several putative cis elements. Here we tested whether the proximal GATA element plays a role in basal and inducible regulation of the hBNP promoter. The hBNP promoter was coupled to a luciferase reporter gene (1818hBNPLuc) and transferred into neonatal ventricular myocytes (NVM), and luciferase activity was measured as an index of hBNP promoter activity. Mutation of the putative GATA element at −85 of the hBNP promoter [1818(mGATA)hBNPLuc] reduced activity by 97%. To study transactivation of the hBNP promoter, we co-transfected 1818hBNPLuc with the GATA-4 expression vector. GATA-4 activated 1818hBNPLuc, and this effect was eliminated by mutation of the proximal GATA element. Electrophoretic mobility shift assay showed that an oligonucleotide containing the hBNP GATA motif bound to cardiomyocyte nuclear protein, which was competed for by a consensus GATA oligonucleotide but not a mutated hBNP GATA element. The β-adrenergic agonist isoproterenol and its second messenger cAMP stimulated hBNP promoter activity and binding of nuclear protein to the proximal GATA element. Thus the GATA element in the proximal hBNP promoter is involved in both basal and inducible transcriptional regulation in cardiac myocytes.

scholarly journals PGE2stimulates human brain natriuretic peptide expression via EP4and p42/44 MAPK

2006 ◽  
Vol 290 (5) ◽  
pp. H1740-H1746 ◽  
Author(s):  
Jian-Yong Qian ◽  
Alicia Leung ◽  
Pamela Harding ◽  
Margot C. LaPointe
Keyword(s):  
Protein Synthesis ◽  
Brain Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Promoter Activity ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Dominant Negative Mutant ◽  
Prostaglandin E ◽  
Stimulation Of

Brain natriuretic peptide (BNP) produced by cardiac myocytes has antifibrotic and antigrowth properties and is a marker of cardiac hypertrophy. We previously showed that prostaglandin E2(PGE2) is the main prostaglandin produced in myocytes treated with proinflammatory stimuli and stimulates protein synthesis by binding to its EP4receptor. We hypothesized that PGE2, acting through EP4, also regulates BNP gene expression. We transfected neonatal ventricular myocytes with a plasmid encoding the human BNP (hBNP) promoter driving expression of a luciferase reporter gene. PGE2increased hBNP promoter activity 3.5-fold. An EP4antagonist reduced the stimulatory effect of PGE2but not an EP1antagonist. Because EP4signaling can involve adenylate cyclase, cAMP, and protein kinase A (PKA), we tested the effect of H-89, a PKA inhibitor, on PGE2stimulation of the hBNP promoter. H-89 at 5 μM decreased PGE2stimulation of BNP promoter activity by 100%. Because p42/44 MAPK mediates the effect of PGE2on protein synthesis, we also examined the role of MAPKs in the regulation of BNP promoter activity. PGE2stimulation of the hBNP promoter was inhibited by a MEK1/2 inhibitor and a dominant-negative mutant of Raf, indicating that p42/44 MAPK was involved. In contrast, neither a p38 MAPK inhibitor nor a JNK inhibitor reduced the stimulatory effect of PGE2. Involvement of small GTPases was also studied. Dominant-negative Rap inhibited PGE2stimulation of the hBNP promoter, but dominant-negative Ras did not. We concluded that PGE2stimulates the BNP promoter mainly via EP4, PKA, Rap, and p42/44 MAPK.

scholarly journals Hormonal regulation of the human sterol 27-hydroxylase gene CYP27A1

2003 ◽  
Vol 372 (2) ◽  
pp. 529-534 ◽  
Author(s):  
Zufan ARAYA ◽  
Wanjin TANG ◽  
Kjell WIKVALL
Keyword(s):  
Reporter Gene ◽  
Hormonal Regulation ◽  
Promoter Activity ◽  
Luciferase Activity ◽  
Luciferase Reporter ◽  
Signal Transduction Pathways ◽  
Cytochrome P450 Enzyme ◽  
Luciferase Reporter Gene ◽  
Response Elements ◽  
P450 Enzyme

The mitochondrial sterol 27-hydroxylase (CYP27A1) is a multifunctional cytochrome P450 enzyme that catalyses important hydroxylations in the biosynthesis of bile acids and bioactivation of vitamin D3. Previous results [Babiker, Andersson, Lund, Xiu, Deeb, Reshef, Leitersdorf, Diczfalusy and Björkhem (1997) J. Biol. Chem. 272, 26253–26261] suggest that CYP27A1 plays an important role in cholesterol homoeostasis and affects atherogenesis. In the present study, the regulation of the human CYP27A1 gene by growth hormone (GH), insulin-like growth factor-1 (IGF-1), dexamethasone, thyroid hormones and PMA was studied. HepG2 cells were transfected transiently with luciferase reporter gene constructs containing DNA fragments flanking the 5′-region of the human CYP27A1 gene. GH, IGF-1 and dexamethasone increased the promoter activity by 2–3-fold, whereas thyroxine (T4) and PMA repressed the activity significantly when measured with luciferase activity expressed in the cells. The endogenous CYP27A1 enzyme activity in the cells was stimulated by GH, IGF-1 and dexamethasone, whereas T4 and PMA inhibited the activity. Experiments with progressive deletion/luciferase reporter gene constructs indicated that the response elements for GH may be localized in a region upstream to position −1094 bp. The putative response elements for dexamethasone were mapped to positions between −792 and −1095 bp. The −451 bp fragment of the human CYP27A1 gene was found to confer the activation by IGF-1, and the inhibition by T4 and PMA. Results of the present study suggest that CYP27A1 is regulated in human cells by hormones and signal-transduction pathways.

scholarly journals Regulation of the Rat Follicle-Stimulating Hormone β-Subunit Promoter by Activin

2003 ◽  
Vol 17 (3) ◽  
pp. 318-332 ◽  
Author(s):  
Magdalena I. Suszko ◽  
Denise J. Lo ◽  
Hoonkyo Suh ◽  
Sally A. Camper ◽  
Teresa K. Woodruff
Keyword(s):  
Promoter Activity ◽  
Hormone Secretion ◽  
Signal Transduction Pathway ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Luciferase Reporter Gene ◽  
Additional Increase ◽  
Smad Binding Element ◽  
Stimulation Of

Abstract FSH is controlled by a variety of positive and negative stimuli, and the unique FSHβ-subunit is a major target for this regulation. Activin is a key modulator of FSHβ transcription and hormone secretion. The signal transduction pathway leading to FSH expression was previously unknown. Here, we show that the transcription factors Smad3 and Smad4 mediate activin-stimulated activity of the rat FSHβ promoter in a pituitary-derived cell line, LβT2. Cells were transiently transfected with the rat FSHβ promoter fused to a luciferase reporter gene (−338rFSHβ-Luc), and a minimal activin-responsive region was identified. Transfection of Smad3, but not the highly related Smad2, led to a ligand-independent stimulation of the FSHβ promoter activity. As expected, activin caused an additional increase of luciferase expression, which was blocked by cotreatment with follistatin. Although Smad4 alone had no effect on FSHβ transcription, it significantly augmented Smad3 and activin-mediated stimulation of the promoter. A palindromic consensus Smad-binding element in the proximal promoter was found to bind Smad4, and elimination of the region resulted in a loss of activin-mediated FSHβ transcription. The activin signaling pathway is conserved in a number of cells, but FSHβ expression is restricted to gonadotropes. A pituitary-specific transcription factor necessary for activin-dependent induction of the FSHβ promoter has been identified that permits FSHβ expression in nongonadotrope cells. Pitx2 is a member of Pitx subfamily of bicoid-related homeodomain factors that is required for pituitary development and is present in the adult pituitary. This factor was transfected into LβT2 cells, where it caused up-regulation of basal and activin-mediated FSHβ promoter activity. Furthermore, cotransfection of Pitx2c with Smad3 in kidney-derived TSA cells resulted in activin-regulated FSHβ response, suggesting its important role in tissue-restricted regulation of FSHβ by activin. A Pitx2c binding site was identified within the proximal promoter, and elimination of this region also resulted in a loss of activin-regulated FSHβ promoter activity. Taken together, these studies suggest that the regulation of FSHβ is dependent on activin-mediated signaling factors in concert with pituitary-derived nuclear regulatory proteins.

Inducible regulation of human brain natriuretic peptide promoter in transgenic mice

2001 ◽  
Vol 280 (1) ◽  
pp. H368-H376 ◽  
Author(s):  
Quan He ◽  
Ding Wang ◽  
Xiao-Ping Yang ◽  
Oscar A. Carretero ◽  
Margot C. LaPointe
Keyword(s):  
Transgenic Mice ◽  
Brain Natriuretic Peptide ◽  
Cardiac Myocytes ◽  
Natriuretic Peptide ◽  
Luciferase Activity ◽  
Left Ventricular ◽  
Neonatal Rat ◽  
Luciferase Reporter ◽  
Coronary Artery Ligation

Studies have shown that brain natriuretic peptide (BNP) gene expression is rapidly induced in the infarcted heart and that plasma BNP levels reflect the degree of left ventricular dysfunction. Our previous in vitro work using transiently transfected neonatal rat cardiac myocytes has shown that the human BNP (hBNP) promoter, in particular a region extending from −127 to −40 relative to the start site of transcription, is more active in cardiac myocytes than in fibroblasts. To study tissue-specific and transcriptional regulation of the hBNP gene in vivo, we generated transgenic mice containing the proximal hBNP promoter (−408 to +100) coupled to a luciferase reporter gene. In four lines of transgenic mice, luciferase activity was ∼33- to 100-fold higher in the heart than in other tissues, including the whole brain. To test whether the transgene responded to a pathophysiological stimulus, we induced infarction by coronary artery ligation. Luciferase activity was fivefold higher in the infarcted region of the left ventricle at 48 h than in sham-operated animals and remained elevated for 4 wk. Endogenous BNP mRNA was similarly increased in the infarcted hearts of a separate group of mice. We conclude that 1) the proximal 408-bp region of the hBNP promoter confers cardiac-specific expression and 2) myocardial infarction activates the proximal hBNP promoter in vivo. These data suggest that we have a valid model for the study of basal and inducible regulation of the hBNP gene in vivo.

scholarly journals Regulation of Plasminogen Gene Expression by Interleukin-6

Blood ◽  
1997 ◽  
Vol 89 (7) ◽  
pp. 2394-2403 ◽  
Author(s):  
G. Ronald Jenkins ◽  
Dietmar Seiffert ◽  
Robert J. Parmer ◽  
Lindsey A. Miles
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Interleukin 6 ◽  
Luciferase Activity ◽  
Hepatoma Cells ◽  
Luciferase Reporter ◽  
Plasmin Activity ◽  
Luciferase Reporter Gene ◽  
Human Hepatoma Cells

Abstract Plasmin, the primary fibrinolytic enzyme, has a broad substrate spectrum and participates in other biological processes dependent upon proteolytic activity. Consequently, plasmin activity is tightly regulated by plasminogen activators and protease inhibitors. In this study, we examined whether regulation of plasminogen gene expression also might provide a new mechanism for controlling this system. We examined the effects of recombinant human interleukin-6 (rhIL-6), a pleiotropic cytokine, on plasminogen mRNA expression in primary murine hepatocytes and Hep3B human hepatoma cells. In primary hepatocytes, rhIL-6 and hydrocortisone separately increased plasminogen mRNA expression, but hydrocortisone did not markedly enhance the response to rhIL-6. Hep3B hepatoma cells exhibited more modest responses to rhIL-6. We used the polymerase chain reaction to amplify a 1,067-bp fragment of the human plasminogen promoter/5′ flanking region. This fragment was cloned upstream of a luciferase reporter gene. Hep3B cells transiently transfected with this construct provided ∼100-fold higher luciferase activity compared to cells transfected with control plasmids, and luciferase activity was increased ∼4.5-fold when these cells were treated with rhIL-6. Furthermore, mice injected with rhIL-6 exhibited increases in hepatic plasminogen mRNA. Circulating plasminogen levels were significantly higher in the mice injected with rhIL-6 compared to mice injected with saline. Mice injected with lipopolysaccharide (an inducer of IL-6 in vivo) also showed increased hepatic plasminogen mRNA. Thus, plasminogen gene expression can be modulated by rhIL-6, suggesting a new mechanism for regulating biological systems that use plasmin.

scholarly journals Differential regulation of the TRH gene promoter by triiodothyronine and dexamethasone in pancreatic islets

2001 ◽  
Vol 170 (1) ◽  
pp. 91-98 ◽  
Author(s):  
P Fragner ◽  
SL Lee ◽  
S Aratan de Leon
Keyword(s):  
Gene Expression ◽  
Pancreatic Islets ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Flanking Sequence ◽  
Luciferase Reporter Gene ◽  
Neoplastic Cells

TRH was initially found in the hypothalamus and regulates TSH secretion. TRH is also produced by insulin-containing beta-cells. Endogenous TRH positively regulates glucagon secretion and attenuates pancreatic exocrine secretion. We have previously shown that triiodothyronine (T(3)) down-regulates pre-pro-TRH gene expression in vivo and in vitro. The present study was designed to determine the initial impact of T(3) on rat TRH gene promoter and to compare this effect with that of dexamethasone (Dex). Primary islet cells and neoplastic cells (HIT T-15 and RIN m5F) were transiently transfected with fragments of the 5'-flanking sequence of TRH fused to the luciferase reporter gene. The persistence of high TRH concentrations in fetal islets in culture, probably due to transactivating factors, allowed us to explore how T(3) and Dex regulate the TRH promoter activity in transfected cells and whether the hormone effect is dependent on the cell type considered. TRH gene promoter activity is inhibited by T(3) in primary but not neoplastic cells and stimulated by Dex in both primary and neoplastic cells of islets. These findings validate previous in vivo and in vitro studies and indicate the transcriptional impact of these hormones on TRH gene expression in the pancreatic islets.

scholarly journals Calcium Dynamics and Resting Transcriptional Activity Regulates Prolactin Gene Expression

Endocrinology ◽  
2002 ◽  
Vol 143 (9) ◽  
pp. 3548-3554 ◽  
Author(s):  
Carlos Villalobos ◽  
Lucía Núñez ◽  
William J. Faught ◽  
David C. Leaumont ◽  
Fredric R. Boockfor ◽  
...  
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Pituitary Cells ◽  
Luciferase Reporter Gene ◽  
Transcriptional Responses ◽  
Prolactin Gene ◽  
Before And After ◽  
Single Living

Abstract Research on the regulation of hormone gene expression by calcium signaling is hampered by the difficulty of monitoring both parameters within the same individual, living cells. Here we achieved concurrent, dynamic measurements of both intracellular Ca2+ concentration ([Ca2+]i) and prolactin (PRL) gene promoter activity in single, living pituitary cells. Cells were transfected with the luciferase reporter gene under control of the PRL promoter and subjected to bioluminescence and fluorescence imaging before and after presentation of TSH-releasing hormone (TRH), a prototypic regulator of PRL secretion and gene expression that induces a transient Ca2+ release, followed by sustained Ca2+ influx. We found that cells displaying specific photonic emissions (i.e. mammotropes) showed heterogeneous calcium and transcriptional responses to TRH. Transcriptionally responsive cells always exhibited a TRH-induced [Ca2+]i increase. In addition, transcriptional responses were related to the rate of Ca2+ entry but not Ca2+ release. Finally, cells lacking transcriptional responses (but showing [Ca2+]i rises) exhibited larger levels of resting PRL promoter activity than transcriptionally responsive cells. Thus, our results suggest that the sustained entry of Ca2+ induced by TRH (but not the Ca2+ release) regulates transcriptional responsiveness. Superimposed on this regulation, the previous, resting PRL promoter activity also controls transcriptional responses.

Signaling mechanisms underlying strain-dependent brain natriuretic peptide gene transcription

2001 ◽  
Vol 79 (8) ◽  
pp. 640-645 ◽  
Author(s):  
Faquan Liang ◽  
Branka Kovacic-Milivojevic ◽  
Songcang Chen ◽  
Junfeng Cui ◽  
Fred Roediger ◽  
...  
Keyword(s):  
Brain Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Gene Transcription ◽  
Promoter Activity ◽  
Cardiac Myocyte ◽  
Mechanical Strain ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Amino Terminal ◽  
Strain Dependent

Activation of brain natriuretic peptide (BNP) gene promoter activity represents one of the earliest and most reliable markers of ventricular cardiac myocyte hypertrophy. We recently demonstrated that mechanical strain increases immunoreactive BNP secretion, steady-state BNP mRNA levels and BNP gene transcriptional activity in neonatal rat myocyte cultures. We have also shown that strain-dependent BNP gene transcription is critically dependent on the functional integrity of a number of integrins (specfically β1, β3, and αvβ5 integrins) present on the surface of cardiac myocytes. When used alone, each of these antibodies resulted in a significant reduction in strain-dependent activation of a transfected hBNP-luciferase reporter and inhibition of a number of signaling pathways that have been linked to stimulation of this reporter (e.g., extracellular signal regulated kinase and c-Jun amino terminal kinase). The present study shows that combinations of these antibodies resulted in further reductions in hBNP gene promoter activity and inhibition of the relevant signaling cascades. These studies provide further support for the importance of integrin-matrix interactions in promoting strain-dependent changes in cardiac myocyte gene transcription.Key words: mechanical strain, brain natriuretic peptide, integrins, mitogen-activated protein kinase, cardiac myocyte.

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